This invention relates to the measurement of cell counts and identification of crystals in synovial fluid samples and more particularly in providing a control reference material for the quality control of the measurements of these cells and the identification of these crystals of interest.
Synovial fluid, often referred to as joint fluid, is a viscous liquid found in joint cavities and supplies nutrients to the cartilages and acts as a lubricant to the surfaces of the frequently moving joints. It is formed as an ultrafiltrate of the plasma across the synovial membranes, into which a mucopolysaccharide containing hyaluronic acid and a small amount of protein is secreted by the cells of the synovial membrane. The chemical composition of the synovial fluid is essentially the same as human plasma except for the high molecular weight proteins which are normally absent in the synovial fluid. It is established that total cell count, differential white blood cell count and crystal identification on synovial fluid can provide valuable information regarding infection, inflammation, and irritation of the joint spaces. Total cell counts are usually performed in a counting chamber, while crystals are analyzed using light and polarized light microscopy. Red blood cells are seen in synovial fluid samples either as a result of a traumatic tap of the joint or as a result of a hemorrhagic condition. Lymphocytes and other white blood cells are seen during conditions of nonseptic inflammation while neutrophils are seen during conditions of bacterial sepsis and crystal induced inflammation. Identifying the causative crystals in the synovial fluid, herein also referred to simply as fluid, especially if they are found intracellularly in neutrophils and macrophages, is pathognomic for a crystal induced arthritis. Pathognomic means characteristic of a disease condition or stage.
Gout and pseudogout comprise the two major crystal induced arthritides. The presence of intracellular monosodium urate (MSU, hereinafter) crystals in neutrophils and macrophages is found in 90% of patients having an acute attack of gout. These crystals, however, are also seen in synovial fluid between attacks in 75% of patients, suggesting that multiple factors contribute to an acute episode. Pseudogout, or calcium pyrophosphate deposition disease, is characterized by the presence of calcium salts in cartilage and calcium pyrophosphate dihydrate (CPPD, hereinafter) crystals in synovial fluid. Unlike gout, no single serum metabolite is responsible for the disease. Hereditary metabolic problems or endocrine disorders, such as hypothyroidism and hyperparathyroidism, that elevate calcium levels in blood can lead to pseudogout. More commonly, pseudogout is associated with degenerative arthritis, demonstrated by X-ray evidence of articular cartilage calcification. As many as 50% of adults may have CPPD deposits in their joints at the time of their deaths. Techniques for crystal analysis focus on MSU and CPPD, the two most common crystals. MSU crystals are needle shaped and produce negative birefringence with polarized light. CPPD are often rhomboid, but are also seen as needles and rods, and are weakly birefringent with polarized light, producing positive birefringence. The analyses of these crystals using polarized microscopy and the counting of the cells using counting chambers are known. Other birefringent materials that may also be found in the synovial fluid include calcium oxalate, cartilage fibers, collagen, cholesterol, hydroxyapatite, betamethasone acetate, cortisone acetate, methyl prednisone acetate, prednisone tebulate, triamcinolone acetonide, triamcinolone hexacetonide, EDTA (ethylenediamine tetraacetate), fat or cholesterol esters, lithium heparin, starch granules and debris.
Analysis of synovial fluid is widely accepted as a crucial aid in the evaluation of joint diseases. Identification of crystals in joint fluid establishes a definite diagnosis of these diseases and the leukocyte count can be used to broadly group diseases as inflammatory or noninflammatory. Although these analyses are diagnostically crucial, there is no established available reference control material designed to monitor the accuracy, reliability and reproducibility of these tests or analyses. Incidents of misclassification between inflammatory and noninflammatory due to a wide range of cell count reported are known and discrepancies in crystal identification causing false positives and false negatives have been reported. These affect both diagnosis and treatment. Several factors have been proposed as reasons for these errors such as lack of training and experience with the test methodologies by the laboratory analysts, the infrequency of testing performed in synovial fluids, the differences in the quality and nature of the equipment used for performing the tests, the difference in the diluting fluids used for the specimens, the amount of crystals present in a sample or specimen, differences in the viscosity of the samples, procedural variations in the handling and pipetting of the samples, and differences in time after which a sample or specimen is tested after collection. Clearly, quality control measures are needed for these tests.
It is therefore an object of this invention to provide a reference control material that can be easily prepared in commercial quantity to help improve the reliability of the test results obtained by testing laboratories.
It is also an object of this invention to provide a pathognomic reference control material having red and or white blood cells and or crystals for identifying and differentiating patients with degenerative arthritis from those patients suffering from gout.
It is a further object of this invention to provide several diluent or base materials suitable for suspending cell/s and/or crystals for the reference control material.
Laboratories have used synovial fluid obtained from the knee of patients with relatively inactive rheumatoid arthritis as a negative control in the analyses of patients"" synovial fluids. Others have spiked human synovial fluid with known amounts or either monosodium urate (MSU) or calcium pyrophosphate (CPPD) crystals or a combination of these. However, to use human synovial fluid as a base for the preparation of a reference control material is not practical because of its limited quantity. Further, simply spiking the synovial fluid from a normal individual with the crystals or other birefringent materials of interest do not present a pathognomic reference control material because white blood cells and/or red blood cells may not be present in these spiked samples. This invention offers several synovial fluid reference control materials interchangeably referred to herein as synovial fluid control materials of broad flexibility, variety and usage. For a pathognomic synovial fluid reference control material, hereinafter referred to as pathognomic control, it is most preferred to spike a simulated synovial fluid base. This pathognomic control comprises a desired amount of fixed red blood cells, the desired amount less than 5000 cells/ul (microliter), preferably 30-300 cells/ul; a desired amount of fixed white blood cells, the desired amount less than 10000 cells/ul, preferably 300-1500 cells/ul; a desired amount of calcium pyrophosphate crystals, the desired amount less than 300 mg/l (liter), preferably 150 mg/l; and/or a desired amount of sodium urate crystals, the desired amount less than 300 mg/l (liter), preferably 80 mg/l. Because it is a pathognomic control, not all of the components mentioned above need be present in a single formulation but only those required or present in a particular disease state of interest. A simulated synovial fluid base comprises glucose, sodium lactate, potassium and sodium chloride and a preservative. The simulated synovial fluid base preferably contains hyaluronic acid and most preferably contain human serum albumin. The level of cell counts and crystals may vary based on the apparatus and test procedure used to assay the synovial fluid. The above levels or concentrations given herein are preferred simply because they usually need no further dilution before analyses when the most common apparatus or equipment, a hemacytometer and/or a polarizing microscope is/are used.
A method of preparing the pathognomic control in simulated synovial fluid base comprises the steps of: a) dissolving a known amount of glucose, sodium lactate, potassium chloride, sodium chloride and a preservative in water, preferably deionized or distilled; b) stirring the mixture until all added ingredients are in solution; c) adjusting the pH of the solution to 7.0xc2x11.0, preferably 7.4xc2x10.1; d) adding water to the mixture to a known volume; e) filtering the solution through a 0.2 u membrane; f) dividing the solution into two equal parts; g) adding calcium pyrophosphate crystals, white blood cells, and red blood cells to one part; h) adding sodium urate crystals, white blood cells and red blood cells to the other part; i) rechecking the pH of the solution and readjusting the pH to 7.0xc2x11.0, preferably 7.4xc2x10.1 if necessary; j) testing the resulting solutions for appearance, pH, glucose, lactic acid, white blood cell, red blood cell, calcium pyrophosphate crystals and sodium urate crystals; and, k) filling the resulting solutions in vials when the test results are acceptable. To improve the birefringence of the crystals and render viscosity to the pathognomic control, sodium hyaluronate is also added. Human serum albumin is also preferably added to closely resemble the protein concentration of a human synovial fluid. Sodium hyaluronate or hyaluronic acid sodium salt is preferably added to a final concentration of less than 1%, preferably 0.1% by initially dissolving sodium hyaluronate in water prior to the addition of the other ingredients enumerated in step a) above. Human serum albumin, when preferred, is usually added with the other ingredients of step a) to a final concentration of 2xc2x10.2 g/dl.
While the preferred pathognomic control illustrated above uses a simulated synovial fluid base, it is found that normal saline, a 0.9% sodium chloride solution; water, preferably deionized or distilled; or, any buffer solution unreactive to the crystals and cells, can be used as a suspending base. Normal saline, water and buffer solutions with pH 7.0xc2x11.0 are herein collectively referred to as aqueous base. The aqueous base and the simulated synovial fluid base are collectively referred to herein as suspending base. Typical buffer solutions suitable for use are phosphate buffers, barbiturate/HCl, imidazole/HCl, MOPS [3-(N-morpholino) propanesulfonic acid], N-(2-acetimido)-2-imidoacetic acid, PIPES (1,4-Piperazinediethanesulfonic acid) and the like. The crystals, sodium urate and calcium pyrophosphate, preferably a dihydrate, and the cells, white blood cells and red blood cells, are referred to herein as the main components of interest. The respective cations of urate and pyrophosphate and their levels of hydration, if applicable, may vary so long as their shape and solubility remains the same.
A synovial fluid reference control material or synovial fluid control in a suspending base may contain only one main component of interest or may combine several of these components in a common suspending base without any deleterious effect. These synovial reference control materials may be prepared by simply adding one of the main components or the components of choice in the suspending base. Aside from the main components of interest, reference control materials may be prepared for the other birefringent materials that may be found in the synovial fluids such as calcium oxalate, cartilage fibers, collagen, cholesterol, hydroxyapatite, betamethasone acetate, cortisone acetate, methyl prednisone acetate, prednisone tebulate, triamcinolone acetonide, triamcinolone hexacetonide, EDTA (ethylenediamine tetraacetate), fat or cholesterol esters, lithium heparin, starch granules and debris in a similar manner. An aqueous base is preferred over a simulated synovial fluid base when the cost of preparing the synovial fluid control is a major factor.
The normal amount of synovial fluid in the knee cavity is less than 3.5 milliliter (ml). Therefore, it is impractical to prepare a reference control material in a commercial quantity using human synovial fluid as a base. The initial tendency is to formulate a simulated synovial fluid base mimicking the composition of human synovial fluid wherein known amounts of components to be tested can be added which would serve as a control sample. While this is preferred, it was found that equally suitable reference control materials for synovial fluid analyses can be prepared by adding anyone of or any combination of the main components of interest, namely, urate and pyrophosphate crystals, white blood cells and red blood cells, in the following suspending bases such as normal saline (0.9% sodium chloride in water), water preferably deionized or distilled and other buffer preparations such as phosphate buffers, barbiturate/HCl, imidazole/HCl, MOPS [3-(N-morpholino) propanesulfonic acid], N-(2-acetimido)-2-imidoacetic acid, PIPES (1,4-Piperazinediethanesulfonic acid) and the like. This finding is significant because a large volume of synovial fluid reference control material or synovial fluid control can be prepared at a minimal cost. Additionally, it would be just as easy to prepare control materials for the other birefringent materials found in synovial fluid, typical examples of which were enumerated above. The method of preparing these controls has as a main step, the dissolution of any one of or a combination of the main components of interest in a desired suspending base such as water, normal saline, any suitable buffer solution having a pH of 7.0xc2x11.0, preferably at pH 7.4xc2x10.1, or simulated synovial fluid. It is found that adding a combination of some or all of the components together is possible without any deleterious effect. The desired concentration of the component/s to be added to the suspending base is at the discretion of the control manufacturer or the testing laboratory which is usually dictated by the type of equipment or apparatus used for counting the cells and/or the type of equipment or apparatus used for analyzing the presence of the crystals, more particularly its birefringence under polarized light. The typical concentrations which may not require further dilution before analysis are as follows: fixed red blood cells, less than 5000 cells/ul (microliter), preferably 30-300 cells/ul; fixed white blood cells, less than 10000 cells/ul, preferably 300-1500 cells/ul; calcium pyrophosphate crystals, less than 300 mg/l (liter), preferably 150 mg/l; sodium urate crystals, less than 300 mg/l (liter), preferably 80 mg/l.
The ability to suspend the components of interest in an aqueous base was found to be feasible because of the use of fixed white blood cells and fixed red blood cells. Methods of fixing the cells are known and fixed cells are commercially available. The fixed white blood cell and fixed red blood cell used herein come in concentrations of approximately 1010 cells/liter. The pH of the base is important because extreme pH conditions (below 6 and above 8) may affect the solubility of the urate and pyrophosphate crystals and may also lyse the cells. A preservative is usually added to prevent bacterial contamination. The typical concentrations of preservatives depend upon the type of preservative used and are known in the art. Example A shows a method of preparing a one component synovial fluid control in normal saline.
The synovial fluid control described above can have only one or more of the components of interest. However, one may desire a pathognomic synovial fluid reference material which contains all of the main components at concentration ranges covering those typically found in a particular disease condition or state. For a pathognomic control, the main components may be added to an aqueous base or to a simulated synovial fluid base, which are collectively referred to as suspending base. Example B shows the method of preparing a pathognomic control in normal saline. The pathognomic controls may be prepared separately according to a disease state or combined to serve as control for different disease states. The pathognomic controls illustrated in Examples B and C are prepared separately according to a disease state, identified as Type A and Type B. Type A preferably contains calcium pyrophosphate dihydrate crystals, fixed red blood cells and fixed white blood cells, predominantly, lymphocytes. The Type A control is used primarily to detect patients with degenerative arthritis. Calcium pyrophosphate dihydrate crystals appear as rhomboids, in needle or rod form and are weakly birefringent with polarized light, producing a positive birefringence. The Type B control contains sodium urate, fixed red blood cells and fixed white blood cells, predominantly neutrophils. This reference control material is used to detect patients suffering from gout. Sodium urate appear as needles and produce a negative birefringence with polarized light.
Birefringent materials have the ability to bend or refract light due to the presence of a molecular grain produced by the nonrandom, linear internal structure of the crystals, the grain running down the length of the crystal. This molecular grain causes incident light passing through the crystal to split in two rays: a fast ray which passes with the molecular grain, and a slow ray which passes 90 degrees to the fast ray or against the molecular grain. Whether the birefringence is negative or positive is determined by a formula: BR=(RIlxe2x88x92RIxe2x8axa5). The birefringence is positive if the refractive index parallel to the long axis of the crystal is greater than the refractive index perpendicular to the long axis of the crystal. It is negative if the relationship is reversed.
The preparation of synovial fluid control in an aqueous base has been described above. For a pathognomic synovial control in simulated synovial fluid base, as illustrated in Example C, the crystals and fixed white blood cells together with fixed red blood cells are mixed with the simulated synovial fluid base containing glucose, sodium lactate, potassium chloride, and preferably, sodium hyaluronate. Human serum albumin is also preferably added to the simulated synovial fluid base. To prevent bacterial contamination, a preservative is usually added to the reference control material. The addition of sodium hyaluronate is preferred although not strictly necessary, to impart viscosity. It was observed that a viscosity similar to the natural human synovial fluid improved the birefringence of the crystals under polarized light. For a control reference material containing both hyaluronate and human serum albumin, the composition and preferred levels are as follows: human serum albumin, 1-5%, preferably 2%; hyaluronic acid, 0-1%, preferably 0.1%, calcium pyrophosphate, 0-300 mg/l, preferably 150 mg/l; sodium urate needles, 0-300 mg/l, preferably 80 mg/l; glucose, 00.1%, preferably 0.08%; sodium lactate, 0-0.1%, preferably 0.02%, potassium chloride, 0-1%, preferably 0.03%; sodium chloride 0-1%, preferably 0.6% and a preservative. 0-0.5%, preferably 0.2% sodium azide is sufficient. Other polymers commonly known to impart viscosity can be used instead of hyaluronic acid such as dextran, polyethylene glycol, gelatin, starch, gums, polysaccharides and the like. These polymers, however, unlike hyaluronate, may not improve the birefringence of the crystals.
All the examples herein are mainly provided for illustration and not as a limitation to the invention especially with regards to the different suspending bases, the relative concentrations of the components, i.e. birefringent crystals, white blood cells, red blood cells, the relative amounts of the ingredients in the simulated synovial fluid base and the separation of the pathognomic control according to a disease state.